Pressurized Perfusion System for Obtaining Completely Acellular Pulmonary Valve Scaffolds for Tissue Engineering

Abstract Introduction. Xenogeneic tissues decellularization represents the obtaining process of extracellular matrix derived scaffolds. Most antigens being cell based, non-immunogenicity is obtained by cells removal. Scaffolds are temporary structures with biologic and mechanical role. Scaffolds, stem cells and bioreactors represent premise of regenerative medicine, aiming towards the ideal valvular substitute. In previous studies, we decellularized pulmonary valves root by immersion histology revealing cellular residue, requiring a more efficient approach. We hypothesized that immersion is insufficient and thus a pressure gradient was added. Material and Method. This is part of a grant approved by the UMFTS. Eleven porcine pulmonary valves were included in the study: n=6 underwent immersion decellularization and n=5 were cyclically perfused with a 20-25mmHg pressure gradient during a 10-day protocol. The acellular valves obtained underwent a quality control using DAPI (4′,6-diamidino-2-phenylindol) nuclear staining, histological Haematoxylin-Eosin, DNA extraction and quantification, harvested from different structural levels: arterial wall, sinus, cusp. Results. Histological assessments highlighted integrity of extracellular matrix in both groups and overall cells absence at the different levels of valvular structures analyzed. Immersion decellularized valves exhibited DAPI positive structures identified as potential residual nucleic material. Comparatively, the perfusion decellularized valves, lacked in those structures, result confirmed by DNA extraction and quantitation procedure. Conclusions. Perfusion decellularization represents a feasible approach to obtain acellular cardiac valvular scaffolds derived from the extracellular matrix, being superior to immersion decellularization method. Their nonimmunogenic potential is underlined by total absence of nuclei. The process is fast, allowing production of an abundant number of valvular biomaterials in a short time.